Target enrichment of metazoan mitochondrial DNA with hybridization capture probes

Abstract

High-throughput DNA sequencing studies allow rapid and widescale investigations into complex assemblages of organisms that would otherwise remain infeasible. Recently scientists have begun to apply metagenomic sequencing (sequencing of the complete pool of DNA from a sample) to investigate microscopic animal communities. Mitochondrial genomes are an important locus for such studies; however, current databases of complete mitochondrial genomes remain incomplete, and many important meiofaunal clades are underrepresented or missing entirely. This underrepresentation is likely due to the low amount of input DNA acquired from meiofaunal individuals, the impossibility of pooling animals because of high levels of cryptic speciation, and the paucity of researchers collecting and studying these animals. Here we help to alleviate the first two barriers by developing a method of enriching animal mtDNA from next-generation sequencing libraries by using hybridization capture probes. These hybrid-capture probes were designed based on the complete collection of mitochondrial genomes available from GenBank and are suitable for enrichment of diverse animal lineages. We demonstrate the protocol on forty-seven meiofaunal specimens spanning four different phyla. Overall, we observed an average of ~450× fold enrichment over standard NGS sequencing library preparation. In addition, we were able to enrich the mtDNA from taxa distantly related to those in the database, indicating that even animals that were not directly targeted by the probe design can be enriched with this method.